期刊：
Trends in Biotechnology,2005年23(2):92-96 ISSN：0167-7799
通讯作者：
Li, K
作者机构：
[Li, K] Nanhua Univ, N Dist Sch, SNP Inst, Hengyang 421001, Hunan, Peoples R China.;Geomapping Inc, Tianjin, Peoples R China.;City Hope Natl Med Ctr, Dept Human Genet, Duarte, CA 91010 USA.;First Mil Med Univ, Inst Oncol, Guangzhou 510515, Peoples R China.
通讯机构：
[Li, K] N;Nanhua Univ, N Dist Sch, SNP Inst, Hengyang 421001, Hunan, Peoples R China.
摘要：
DNA polymerases with 3'-5' proofreading function mediate high fidelity DNA replication but their application for mutation detection was almost completely neglected before 1998. The obstacle facing the use of exo(+) polymerases for mutation detection could be overcome by primer-3'-termini modification, which has been tested using allele-specific primers with 3' labeling, 3' exonuclease-resistance and 3' dehydroxylation modifications. Accordingly, three new types of single nucleotide polymorphism (SNP) assays have been developed to carry out genome-wide genotyping making use of the fidelity advantage of exo(+) polymerases. Such SNP assays might also provide a novel approach for re-sequencing and de novo sequencing. These new mutation detection assays are widely adaptable to a variety of platforms, including real-time PCR, multi-well plate and microarray technologies. Application of exo(+) polymerases to genetic analysis could accelerate the pace of personalized medicine.
关键词：
onychin;vascular smooth muscle;cell cycle;retinoblastoma;cyclin D1;cyclin E;tyrosine kinase;mitogen-activated protein kinase
摘要：
Aim: To investigate the effects of onychin on the proliferation of cultured rat artery vascular smooth muscle cells (VSMCs) in the presence of 10% new-born calf serum (NCS).Methods: Rat VSMCs were incubated with onychin 150 mol/L or genistein 10 mol/L in the presence of 10% NCS for 24 h. The proliferation of VSMCs was measured by cell counting and MTS/PMS colorimetric assays. Cell cycle progression was evaluated by flow cytometry. Retinoblastoma (Rb) phosphorylation, and expression of cyclin D1 and cyclin E were measured by Western blot assays. The tyrosine phosphorylation of ERK1/2 was examined by immunoprecipitation techniques using anti-phospho-tyrosine antibodies.Results: The proliferation of VSMCs was accelerated significantly in the presence of 10% NCS. Onychin reduced the metabolic rate of MTS and the cell number of VSMCs in the presence of 10% NCS in a dose-dependent manner. Flow cytometry analysis revealed that the G1-phase fraction ratio in the onychin group was higher than that in the 10% NCS group (85.2%vs 70.0%, P< 0.01), while the S-phase fraction ratio in the onychin group was lower than that in 10% NCS group (4.3%vs 16.4%, P< 0.01). Western blot analysis showed that onychin inhibited Rb phosphorylation and reduced the expression of cyclin D1 and cyclin E. The effects of onychin on proliferation, the cell cycle and the expression of cyclins in VSMCs were similar to those of genistein, an inhibitor of tyrosine kinase. Furthermore immunoprecipitation studies showed that both onychin and genistein markedly inhibited the tyrosine phosphorylation of ERK1/2 induced by 10% NCS.Conclusion: Onychin inhibits the proliferation of VSMCs through G1 phase cell cycle arrest by decreasing the tyrosine phosphorylation of ERK1/2, and the expression of cyclin D1 and cyclin E, and sequentially inhibiting Rb phosphorylation.
关键词：
random peptide phage library;vascular endothelial cell;whole-cell screening;cell adhesion;caveolin-1;eNOS
摘要：
Using oxidized low-density lipoprotein (LDL)-injured vascular endothelial cells (ECs) as target cells, peptides specifically binding to the injured ECs were screened from a phage-displaying peptide library by using the whole-cell screening technique after three cycles of the “adsorption-elution-amplification” procedure. Positive phage clones were identified by ELISA, and the inserted amino acid sequences in the displaying peptides were deduced from confirmation with DNA sequencing. The adhesion rate of ECs to monocytes was evaluated by cell counting. The activity of endothelial nitric oxide synthase (eNOS), and the expression levels of caveolin-1 and intercellular adhesion molecule-1 (ICAM-1) were determined by Western blotting. Six positive clones specifically binding to injured ECV304 endothelial cells were selected from fourteen clones. Interestingly, four phages had peptides with tandem leucine, and two of these even shared an identical sequence. Functional analysis demonstrated that the YCPRYVRRKLENELLVL peptide shared by two clones inhibited the expression of ICAM-1, increased nitric oxide concentration in the culture media, and upregulated the expression of caveolin-1 and eNOS. As a result, the adhesion rate of monocytes to ECV304 cells was significantly reduced by 12.1%. These data suggest that the anti-adhesion effect of these novel peptides is related to the regulation of the caveolin-1/nitric oxide signal transduction pathway, and could be of use in potential therapeutic agents against certain cardiovascular diseases initiated by vascular endothelial cell damage.
摘要：
This study examined whether genistein influences the production of nitric oxide (NO) and expression of endothelial nitric oxide synthase (eNOS) and the modulators of eNOS activity in ovariectomized (OVX) rat hearts. Female mature Sprague-Dawley rats were subjected to bilateral ovariectomy, OVX rats were randomly divided into four groups: 17beta-estradiol (0.1 mg/kg, s.c. daily) was used as the positive control; low dose of genistein (0.5 mg/kg, s.c. daily); high dose of genistein (5.0 mg/kg, s.c. daily) and model. Sham operations as controls, the treatment lasted 6 weeks. Blood pressure, heart rate, plasma estradiol, heart and uterine weights were measured. Nitrite production in the myocardium was determined by nitrate reductase method. Protein level of eNOS, caveolin-1 and calmodulin was determined by Western blot. The results showed that nitrite production and eNOS protein in homogenized ventricular tissue was attenuated by approximately 53% and 67% in OVX rats compared with those in sham rats, respectively. Genistein increased nitrite production in rat heart in a dose-dependent manner, genistein at the dose of 5 mg/kg.d(-1) resumed nitrite production to a level similar to that in sham operated rats. Administration of genistein also increased eNOS protein expression in OVX rats myocardium with a concomitant decrease in the expression of caveolin-1, an endogenous eNOS inhibitory protein. Another eNOS stimulatory protein, calmodulin, was unchanged in these treatments. These effects were also observed in rats treated with 17beta-estradiol. Genistein at the dose of 5.0 mg/kg.d(-1) augmented uterine weight but this side effect in reproductive system was less than that of 17beta-estradiol. These results suggest that genistein supplementation and estrogen replacement therapy directly increase eNOS functional activity and NO production in the hearts of the OVX rats, but genistein has less side effects on the reproductive system than 17beta-estradiol.
关键词：
apoptosis;Carrier Proteins;Chlamydia trachomatis;membrane proteins;Proto-Oncogene Proteins;Tumor Suppressor Protein p53
摘要：
We have previously correlated Chlamydia trachomatis antiapoptotic activity with the blockade of mitochondrial cytochrome c release and the inhibition of Bax and Bak activation. We now report that C. trachomatis infection leads to degradation of Bik, Puma, and Bim, three upstream proapoptotic BH3-only proteins of the Bcl-2 family that can transmit death signals to mitochondria by inhibiting the Bcl-2 antiapoptotic proteins and/or activating the Bcl-2 proapoptotic members, such as Bax and Bak. This observation has provided new information on the chlamydial antiapoptosis mechanisms.
期刊：
Russian Journal of Genetics,2005年41(7):755-759 ISSN：1022-7954
通讯作者：
Zheng, JF
作者机构：
[Zheng, JF] Nanhua Univ, Coll Life Sci & Technol, Inst Cell & Genet, Hengyang 421001, Peoples R China.;Nanhua Univ, Coll Med, Inst Physiol, Hengyang 421001, Peoples R China.;Nanhua Univ, Coll Life Sci & Technol, Inst Zool, Hengyang 421001, Peoples R China.
通讯机构：
[Zheng, JF] N;Nanhua Univ, Coll Life Sci & Technol, Inst Cell & Genet, Hengyang 421001, Peoples R China.
关键词：
Rainbow Trout;Homologous Sequence;Amino Acid Identity;Acid Identity;Equivalent Region
摘要：
In order to understand sequence information about turtle HMG1 gene, a cDNA encoding HMG1 protein of the Chinese soft-shell turtle (Pelodiscus sinensis) was amplified by RT-PCR from kidney total RNA, and was cloned, sequenced and analyzed. The results revealed that the open reading frame (ORF) of turtle HMG1 cDNA is 606 bp long. The ORF codifies 202 amino acid residues, from which two DNA-binding domains and one polyacidic region are derived. The DNA-binding domains share higher amino acid identity with homologues sequences of chicken (96.5%) and mammalian (74%) than homologues sequence of rainbow trout (67%). The polyacidic region shows 84.6% amino acid homology with the equivalent region of chicken HMG1 cDNA. Turtle HMG1 protein contains 3 Cys residues located at completely conserved positions. Conservation in sequence and structure suggests that the functions of turtle HMG1 cDNA may be highly conserved during evolution. To our knowledge, this is the first report of HMG1 cDNA sequence in any reptilian.
作者机构：
[Zhang, P] Hunan Normal Univ, Coll Life Sci, Changsha 410081, Peoples R China.;Nanhua Univ, Coll Life Sci & Biotechnol, Hengyang 421001, Peoples R China.
通讯机构：
[Zhang, P] H;Hunan Normal Univ, Coll Life Sci, Changsha 410081, Peoples R China.
关键词：
Amanita exitialis;Amatoxins;HPLC;ITS;MS
摘要：
Amanita exitialis Zhu L. Yang and T.H. Li is a lethal mushroom species recently isolated in Guangdong Province, China. In this report, a pure culture of this species was obtained for the first time. To confirm the identity of the pure culture, the internal transcribed spacer regions of the nuclear ribosomal DNA of the pure culture and of a typical fruiting body of the species were sequenced and compared. Further, amatoxins produced by pure cultures were analyzed and characterized by high-performance liquid chromatography and mass spectrometry analysis. The results showed that the pure cultures produced 728.3 ± 43.8 μg g−1 (dry matter) of α-Amanitin and 60.0 ± 20.7 μg g−1 (dry matter) of β-Amanitin, respectively, a yield which is about 10% of that produced by fruiting bodies.
作者机构：
City Hope Natl Med Ctr, Duarte, CA 91010 USA.;Nanhua Univ, SNP Inst, Hengyang, Peoples R China.;Genomapping Inc, Tianjin, Peoples R China.;[Li, K] City Hope Natl Med Ctr, 1500 E Duarte Rd, Duarte, CA 91010 USA.
通讯机构：
[Li, K] C;City Hope Natl Med Ctr, 1500 E Duarte Rd, Duarte, CA 91010 USA.
摘要：
The role of 3' exonuclease excision in DNA polymerization was evaluated for primer extension using inert allele specific primers with exonuclease-digestible ddNMP at their 3' termini. Efficient primer extension was observed in amplicons where the inert allele specific primers and their corresponding templates were mismatched. However, no primer-extended products were yielded by matched amplicons with inert primers. As a control, polymerase without proofreading activity failed to yield primer-extended products from inert primers regardless of whether the primers and templates were matched or mismatched. These data indicated that activation was undertaken for the inert allele specific primers through mismatch proofreading. Complementary to our previously developed SNP-operated on/off switch, in which DNA polymerization only occurs in matched amplicon, this new mutation detection assay mediated by exo(+) DNA polymerases has immediate applications in SNP analysis independently or in combination of the two assays.
期刊：
American Heart Journal,2005年150(5):1039-1045 ISSN：0002-8703
通讯作者：
Fu, MD
作者机构：
[Fu, MD] Sichuan Univ, W China Med Ctr, Dept Biochem & Mol Biol, Apolipoprot Res Unit, Chengdu 610041, Sichuan, Peoples R China.;Hoist Grp Postdoctoral Work Stn, Chengdu, Sichuan, Peoples R China.;Nanhua Univ, Dept Biochem & Mol Biol, Hengyang, Hunan, Peoples R China.
通讯机构：
[Fu, MD] S;Sichuan Univ, W China Med Ctr, Dept Biochem & Mol Biol, Apolipoprot Res Unit, Chengdu 610041, Sichuan, Peoples R China.
摘要：
Background: To investigate the alterations of high-density lipoprotein (HDL) subclasses in endogenous hypertriglyceridemic subjects. Methods: Apolipoprotein A-I contents of plasma HDL subclasses were quantitated by 2-dimensional gel electrophoresis in 236 normolipidemic subjects (including 146 males and 90 females) and 176 endogenous hypertriglyceridemic subjects (including 103 males and 73 females). Results: Apolipoprotein A-I contents of small-sized pre-β1-HDL and HDL3a were significantly higher (P < .01 and P < .01, respectively), but those of large-sized HDL2a and HDL2b were significantly lower (P < .01 and P < .01, respectively) in hypertriglyceridemic subjects versus normolipidemic subjects. Moreover, with the elevation of triglyceride levels, small-sized pre-β1-HDL and HDL3a increased successively; however, large-sized HDL2a and HDL2b decreased successively. Males had significantly higher apolipoprotein A-I contents of small-sized pre-β1-HDL and HDL3b (P < .05 and P < .05, respectively), but lower contents of large-sized HDL2b (P < .01) than females in both normolipidemic and hypertriglyceridemic subjects. Conclusions: The particle size of HDL shifted toward smaller size in hypertriglyceridemic subjects, especially in male subjects. Of note, the shift was more obvious with the elevation of triglyceride levels. The changes mentioned above indicate that HDL maturation might be abnormal and reverse cholesterol transport might be weakened.
摘要：
To investigate the inhibitory effect of the Bcl-XL small interfering RNA (siRNA) on Bcl-XL gene expression in the human gastric cancer cell line MGC-803, green fluorescent protein (GFP) siRNA was constructed and transfected into MGC-803 cells, together with GFP expression vector pTrace SV40. GFP expression levels were observed using fluorescence microscopy. Bcl-XL siRNA and negative siRNA were then constructed and stably transfected into MGC-803 cells. RT-PCR and immunofluorescence were used to detect the expression of Bcl-XL. Spontaneous apoptosis was detected by acridine orange (AO) and flow cytometry. Results were as follows: (1) 48 h after GFP expression vector and GFP siRNA co-transfection, the expression level of GFP in the GFP siRNA group was much lower than the negative siRNA group, according to fluorescence microscopy results. The mRNA and protein levels of Bcl-XL in Bcl-XL siRNA stable transfectants were reduced to almost background level compared with negative siRNA transfectants or untreated cells. (2) Changes in nucleus morphology was observed by AO staining nucleic and flow cytometry analysis, which showed that stable Bcl-XL siRNA transfectants have an increased spontaneous apoptosis (21.17%±1.26% vs. 1.19%±0.18% and 1.56%±0.15% respectively, P<0.05 vs. negative siRNA or untreated control). siRNA targeting GFP or Bcl-XL genes can specifically suppress GFP or Bcl-XL expression in MGC-803 cells, and Bcl-XL siRNA can increase spontaneous apoptosis. Bcl-XL siRNA may be a beneficial agent against human gastric adenocarcinoma.
摘要：
Type 2 diabetes is a major risk factor of the development of atherosclerosis in humans. However, studies examining mechanisms underlying diabetes-accelerated atherosclerosis have been limited by the lack of suitable humanoid animal models. Pigs have a cardiovascular system that is very similar to that of humans and is useful as a model for human physiology and pathophysiology. In this study, we established a new miniature pig model for studying dyslipidaemia and atherosclerosis in diabetes. Chinese Guizhou minipigs were fed a normal control diet or a high-fat/high-sucrose diet (HFSD) for 6 months. Plasma total cholesterol (TC), high-density lipoprotein cholesterol, triglyceride (TG), insulin and glucose were quantified at monthly intervals. The induction of insulin resistance and dysfunction of the pancreatic β-cell were assessed by oral glucose tolerance test and insulin sensitivity test. The aortic fatty streak lesions were quantified following lipid staining with Sudan IV. During the feeding period, mild high plasma TC and TG were induced. At the end of 6 months, in HFSD-fed animals, the adipocytes were hypertrophic, fat deposit in the liver was observed, loss of pancreatic β-cells was observed, and the aortic fatty streak lesions were clearly present in the animals' aortas. Our study established that miniature pigs that were fed a HFSD without adding dietary cholesterol developed insulin resistance, mild diabetes and atherosclerotic lesions. HFSD-fed miniature pigs may be good animal models for research on the treatment of diabetic dyslipidaemia complicated with atherosclerosis.
期刊：
JOURNAL OF ENDOCRINOLOGY,2004年180(3):399-408 ISSN：0022-0795
通讯作者：
Yin, W
作者机构：
[Yin, W] Nanhua Univ, Sch Life Sci & Technol, Dept Biochem & Mol Biol, Hunan 421001, Peoples R China.;Nanhua Univ, Sch Life Sci & Technol, Dept Biochem & Biotechnol, Hunan 421001, Peoples R China.;Cent S Univ, Xiangya Med Coll, Dept Pathophysiol, Changsha, Hunan, Peoples R China.;Otsuka Pharmaceut Fact Inc, Res & Dev, Tokushima, Japan.;Univ Tsukuba, Inst Basic Med Sci, Dept Pathol, Lab Cardiovasc Dis, Tsukuba, Ibaraki 3058575, Japan.
通讯机构：
[Yin, W] N;Nanhua Univ, Sch Life Sci & Technol, Dept Biochem & Mol Biol, Hunan 421001, Peoples R China.
摘要：
The synthetic compound NO-1886 (ibrolipim) is a lipoprotein lipase activator that has been proven to be highly effective in lowering plasma triglycerides. Recently, we found that NO-1886 also reduced plasma free fatty acids and glucose in high-fat/high-sucrose diet-induced diabetic rabbits. In the current study, we investigated the effects of NO-1886 treatment on ectopic lipid deposition and the islet pathology in miniature swine fed a high-fat/high-sucrose diet. Our results showed that feeding this diet to miniature swine caused insulin resistance, increased lipid deposition in non-adipose tissue, such as in the heart, skeletal muscle, liver and pancreas, and also caused pancreatic beta cell damage. However, supplementing 1% NO-1886 (200 mg/kg per day) into the high-fat/high-sucrose diet decreased ectopic lipid deposition, improved insulin resistance, and alleviated the beta cell damage. These results suggest that improvement of lipid disorder, non-adipose tissue steatosis and insulin resistance may be very important for the protection of beta cell damage. Therefore, NO-1886 is potentially beneficial for the treatment of insulin-resistance syndrome.
